Project Summary Disruption of the defense barriers between the host and its indigenous microflora leads to opportunistic infections which can have a serious impact on human health. Intra-abdominal abscesses are formed following perforation of the bowel often due to appendicitis, diverticulitis, carcinoma, or surgery. These infections are composed of mixed intestinal flora with the indigenous intestinal anaerobe, Bacteroides fragilis as the predominant component. We hypothesized that resistance to oxidative stress is an important factor in the development of these infections. This is because relative to the colon, the peritoneal cavity is an oxygenated environment, and the recruitment of PMNs to the site of infection will result in exposure of B. fragilis to reactive oxygen species. In the current funding period we have documented that there is an acute oxidative stress response which is designed to minimize the immediate affects of oxygen radicals. This is mediated by the regulator OxyR which is necessary for abscess formation in mice. We also have shown that there is a novel, widespread induction of genes associated with metabolism, which occurs when there is extended exposure to oxidative stress. In this application, we hypothesize that this novel,expansive induction of metabolic genes is critical to in vivo survival of these anaerobes in extra-intestinal sites. We will determine that this metabolicgene response(MGR)isnecessarytomaintaintheorganismsinaresistantstate,replenishreducing equivalents and energy, and ultimately it enhances persistence in the abscess milieu of necrotic cell debris, viable PMNs, and host serum factors. Three specific aims will address this central hypothesis. In specific aim 1 we will elucidate the mechanisms that regulate the widespread MGR, determine how it is controlled by stress stimuli, and if it leads to resistance to multiple stresses. In specific aim 2 we will show that a robust thiol metabolism is an integral component of the MGR and determine if it is needed to survive in vivo. Finally in specific Aim 3 we will examine mechanisms of iron storage and how they help prevent oxidative damage during the MGR. We will determine if the iron storage proteins can contribute to the repair of iron sulfur proteins and the in vivo expression of their cognate genes will be measured to gain insight into their role during infection.